Ppar-gamma Mediated Transcriptional Regulation

Reporter constructs, pIDE and pIDE_ΔP, were generated by ligating varying lengths of IDE upstream sequence into the pGL₃-Basic luciferase expression vector (Hanheng Biotechnology Co., Shanghai, China) (Supplementary Figure 1). Lipofectamine 3000 Reagent (Invitrogen Life Technologies, California, USA) was used to transfect reporter constructs into HK-2 cells, as described in our previous report.^{[29 (link)]} At about 6 h post-transfection, the same amount of complete medium was added and then the cells were treated with 10 μM rosiglitazone, a selective PPARγ agonist (Cayman Chemical, Ann Arbor, Michigan), 5 μM GW9662, a PPARγ antagonist (Cayman Chemical, Ann Arbor, Michigan), or solvent for 42 h. Firefly and Renilla luciferase activities were measured using the Dual Luciferase Kit (Beyotime, Shanghai, China). The firefly luciferase values of each sample were normalized by Renilla luciferase activity and reported as relative light units.